Antonio Antonucci

Detection of 2H-1,4-thiazine-5,6-dihydro-3-car☐ylic acid (aminoethylcysteine ketimine) in the bovine brain

2H-1,4-Thiazine-5,6-dihydro-3-carboxylic acid (trivial name: aminoethylcysteine ketimine) is a cyclic sulfur-containing imino acid detected in bovine brain extracts by means of three different procedures. Gas liquid chromatography of protein-free extracts of five bovine brains revealed the presence of this compound at concentrations ranging from 2 to 3 nmol/g wet weight of tissue. The enzymatic method based on the inhibition of D-amino acid oxidase activity by aminoethylcysteine ketimine together with an high-performance liquid chromatography procedure confirm the identification and quantitations obtained with gas liquid chromatography. The discovery of this compound structurally similar to pipecolic acid opens the question of its physiological role in the central nervous system.

Detection of Cystathionine Ketimine and Lanthionine Ketimine in Human Brain

The sulfur containing imino acids cystathionine ketimine (CK) and lanthionine ketimine (LK) have been detected in the human brain by an HPLC procedure. The HPLC procedure takes advantage of the selective absorbance at 380 nm of the phenylisothiocyanate-ketimine adduct. Quantitation of cystathionine ketimine and lanthionine ketimine indicates a mean concentration (mean ± SD, n = 4) of 2.3 ± 0.8 nmol/g for CK and of 1.1 ± 0.3 nmol/g for LK in four human cerebral cortex samples of neurosurgical source. The identification of these cyclic ketimine derivatives of L-cystathionine and L-lanthionine as normal human metabolites in human nervous tissue may have interesting metabolic and physiological implications.

Detection of Cystathionine Ketimine in Bovine Cerebellum

Abstract: A new sulfur‐containing cyclic imino acid, cystathionine ketimine, has been detected in bovine cerebellum by gas chromatography, gas chromatography‐mass spectrometry, and high pressure liquid chromatography procedures. Gas chromatography and gas‐mass analyses are based on derivatization of endogenous cystathionine ketimine with diazomethane after a simple enrichment procedure. The high pressure liquid chromatography procedure takes advantage of the selective absorbance at 380 nm of the phenyl isothio‐cyanate‐ketimine interaction product. The concentration of this new sulfur imino acid found in a pool of four bovine cerebella is ∼0.5 nmol/g.

[35S]Lanthionine ketimine binding to bovine brain membranes

2H-1,4-Thiazine-5,6-dihydro-3,5-dicarboxylic acid (trivial name: lanthionine ketimine) is a cyclic sulfur-containing imino acid detected in bovine brain extracts. This compound has been synthesized in a heavily labeled form starting from L-[35S]cysteine and purified by high performance liquid chromatography. We demonstrate the existence of a saturable and reversible binding of [35S]lanthionine ketimine to bovine brain membranes. A single population of binding sites with a concentration of 260 +/- 12 fmol/mg protein and a dissociation constant of 58 +/- 14 nM is present. Specific binding is competitively inhibited by other structurally similar imino acids, namely S-aminoethyl-L-cysteine ketimine and cystathionine ketimine. These results suggest a possible functional role for these ketimines in nervous system.

The specificity of cysteamine oxygenase

Oxidation of cysteamine to hypotaurine is catalyzed by cysteamine oxygenase (EC 1.13.11.19), a non-heme iron enzyme of large distribution [l-3] . A similar oxidation of cysteine to cysteine sulfinic acid has been reported to require a different oxygenase [4,.5]. Specificity of cysteamine oxygenase has been studied earlier in this laboratory by assaying a limited number of sulfhydryl-containing compounds. The enzyme was found to be specific for cysteamine under the conditions used in this work [2]. In the present paper we report the results of a more thorough investigation on the oxidation of various sulfhydryl-containing, and one selenhydryl-containing, compounds aimed at establishing the specificity of this oxygenase.

The oxidation of aminoethylcysteine ketimine dimer by oxygen reactive species.

SummaryThe prominent spontaneous reaction of aminoethylcysteine ketimine in the neutral pH range is the concentration-dependent dimerization (Hermann, 1961). The carboxylated dimer first produced loses the free carboxyl yielding the more stable decarboxylated dimer (named simply the dimer in this note). In the search for a possible biochemical activity of this uncommon tricyclic compound we have assayed whether it could interact with oxygen reactive species (H2O2, O2−,•OH) thus exhibiting a scavenging effect of possible biomedical interest. The dimer interacts with H2O2 producing compounds detectable by chromatographic procedures. The presence of Fe2+ stimulates the oxidative reaction by yielding the hydroxyl radical (the Fenton reaction). Using the system xanthine oxidase-xanthine as superoxide producer, the dimer oxidation by O2− has also been documented. Among the oxidation products the presence of taurine and cysteic acid has been established. Identification of remaining oxidation products and investigation of the possible function of the dimer as a biological scavenger of oxygen reactive species are now oncoming.

Effect of aminoethylcysteine ketimine decarboxylated dimer, a natural sulfur compound present in human plasma, on tert-butyl hydroperoxide-induced oxidative stress in human monocytic U937 cells

Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD) is a natural sulphur compound present in human plasma and urine and in mammalian brain. Recently, it has been detected in many common dietary vegetables. The aim of the present study was to evaluate the ability of AECK-DD to affect cellular response of U937 human monocytic cells to tert-butyl hydroperoxide-induced oxidative stress. AECK-DD was incorporated into cells, as confirmed by GC-MS analyses, without any cytotoxic effect. A 24 h treatment with 50 and 250 μM AECK-DD resulted in the incorporation of 0.10±0.01 and 0.47±0.08 ng AECK-DD × 106 cells, respectively. U937 cells pretreated with AECK-DD (in the range 4–100 μM) showed an increased resistance to tert-butyl hydroperoxide-induced necrotic death, as revealed by a higher percent of survival measured at all incubation times with respect to control cells. Moreover, the protective effect exhibited by AECK-DD is significantly stronger with respect to that obtained with other common antioxidants (N-acetyl cysteine and trolox) and comparable, although somewhat higher, to that of vitamin E. This effect seems to be due to the ability of AECK-DD to reduce glutathione depletion and to inhibit lipid peroxidation during tert-butyl hydroperoxide treatment. It can be concluded that AECK-DD protects cultured human monocytic cells against tert-butyl hydroperoxide-induced oxidative stress and subsequent cell death, likely through an antioxidant action inside the cell. Due to its presence in both human plasma and urine, AECK-DD may play a role in the modulation of oxidative processes in vivo.

Identification of aminoethylcysteine ketimine decarboxylated dimer in human plasma

Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD) is a natural sulfur-containing tricyclic compound detected, until now, in human urine and bovine cerebellum. Recently, the antioxidant properties of this compound, and particularly its protective effect on the in vitro oxidation of low-density lipoproteins, have been demonstrated. In this paper, the identification of AECK-DD in human plasma by means of gas chromatography, high-performance liquid chromatography and gas chromatography-mass spectrometry, performed after a simple and fast purification procedure, is reported.

Methylene blue photosensitized oxidation of hypotaurine in the presence of azide generates reactive nitrogen species: formation of nitrotyrosine.

In our previous study on the hypotaurine (HTAU) oxidation by methylene blue (MB) photochemically generated singlet oxygen (1O2) we found that azide, usually used as 1O2 quencher, produced, instead, an evident enhancing effect on the oxidation rate [L. Pecci, M. Costa, G. Montefoschi, A. Antonucci, D. Cavallini, Biochem. Biophys. Res. Commun. 254 (1999) 661-665]. We show here that this effect is strongly dependent on pH, with a maximum at approximately pH 5.7. When the MB photochemical system containing HTAU and azide was performed in the presence of tyrosine, 3-nitrotyrosine was produced with maximum yield at pH 5.7, suggesting that azide, by the combined action of HTAU and singlet oxygen, generates nitrogen species which contribute to tyrosine nitration. In addition to HTAU, cysteine sulfinic acid, and sulfite were found to induce the formation of 3-nitrotyrosine. No detectable tyrosine nitration was observed using taurine, the oxidation product of HTAU, or thiol compounds such as cysteine and glutathione. It is shown that during the MB photooxidation of HTAU in the presence of azide, nitrite, and nitrate are produced. Evidences are presented, indicating that nitrite represents the nitrogen species involved in the production of 3-nitrotyrosine. A possible mechanism accounting for the enhancing effect of azide on the photochemical oxidation of HTAU and the production of nitrogen species is proposed.

Identification of an oxidation product of aminoethylcysteine ketimine dimer

Summary. In continuation of our previous work dedicated to the detection of the oxidation products of aminoethylcysteine ketimine dimer by oxygen reactive species, we give here data for the identification of the α, β unsaturated sulfoxide as the main product of interaction of the dimer with H2O2. Identification has been done on the basis of mass spectrometry and NMR analyses of the product isolated by preparative chromatography.